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. 2018 Nov 7;9:4663. doi: 10.1038/s41467-018-06889-5

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Establishment and characterization of NPC43 cell line. a Histological examination of NPC tissue from patient 43. Although a low tumor content identified, H&E and EBER ISH staining showed the presence of undifferentiated and EBV+ve cancer cells in the tissue. Scale bars, 100 μm. b The phase-contrast micrograph shows the outgrowth of epithelial cells from the explanted NPC tissues from patient 43 at Day 7. Scale bars, 200 μm. c Growth curve of NPC43 cell line. The growth of NPC43 cells from the NPC explants was slow in the beginning and took about 50 days to become confluent for the first two passages (1:2 splitting ratio). After PD 22, the NPC43 cells could be passaged at a 1:4 splitting ratio. The mean doubling time of NPC43 cells was about 8, 4 and 2.5 days at PD 22, 90 and 200, respectively. d NPC43 cells were stained for the expression of cytokeratin AE1/AE3 (green). The nuclei were revealed by Hoechst stain (blue). The epithelial nature of NPC43 was confirmed by the positive staining of pan-cytokeratin in cells using specific antibodies. Scale bar, 20 μm. e Images of EBV FISH revealing the presence of EBV in NPC43 as punctate red dots. Scale bar, 20 μm. f Expression of EBV genes was detected in NPC43 by real-time PCR. C666-1 was used as positive control. Data are shown as mean ± SD from three independent experiments. g Left panel: Representative image of NPC43 tumors subcutaneously grown in NOD/SCID mouse. Tumors were harvested for histological examination 96 days after injection of NPC43 cells. Middle panel: H&E staining shows the presence of undifferentiated tumor cells. Right panel: EBER ISH confirmed that these tumor cells were EBV+ve. Scale bars, 100 μm. h A representative spectral karyotype of a metaphase spread prepared from NPC43 (PD 24). The karyotype of NPC43 exhibits complex chromosomal abnormalities. Karyotype description: 38,X,-Y,i(1)(q10),-3,-3, der(4)t(4;8)(q21;q?), der(5)t(5;11)(q35;?)t(11;12)(?;?),i(5)(p10),i(6)(q10),der(7)t(7;20)(q11;?)t(3;20)(q13;?),der(7)t(3;7)(?;q11),-8,del(9)(p11),del(11)(q11), del(11)(p11),der(12)t(3;12)(?;q13), -13,-14,der(14)t(7;14)(q?;q21),-16,der(17)t(16;17)(q10;q11)t(12;16)(?;?),+19,der(20)t(12;20)(q?;q11)t(3;12)(?;q13),-21,der(21)t(12;21)(p11,p11),-22, der(1;22)(?;p11) [cp12]